Synthetic gene circuits are carefully designed to assemble functionally genetic regulatory devices and implement certain functions through sensing, integration and processing of molecular information in cells. Various synthetic gene circuits have been developed to achieve customizable, programmable functions in cells, including dynamic behaviours, switches and memory, inter-cellular communication, adaptability, cell polarization, digital and analog computation and complex biosynthetic pathways. Most of these gene circuits are constructed by using limited genetic elements and costly, inefficient “trial-error” methods. Therefore, to simplify the design and optimize the sophisticated operation of living cells, the development of a large-scale, functionally well-defined synthetic genetic element library and corresponding computation model and simulation method is very needed.
In the research field of synthetic biology of mammals, engineered synthesis of transcriptional activators and repressors is an important goal in supporting the design of extensible gene circuits. At present, a common strategy for constructing mammalian/eukaryotic transcriptional repressors is to fuse a transcriptional repression domain and an engineered DNA-binding protein domain, such as zinc finger protein, transcription activator-like effector (TALE) and deactivated Cas9 (dCas9) nuclease in the RNA-guided CRISPR (clustered regularly interspaced short palindromic repeats) System. However, transcriptional repression domains, such as the Krüppel-associated box (KRAB) transcriptional repression domain and the mSin interaction domain (SID4), often result in epigenetic modifications nearby the target promoter and thus have a slow response to time. Thus, such transcriptional repression is not suitable for constructing fast-responded and reversible gene circuits.
Another transcription repression mode generally present in prokaryotes is through steric hindrance of nonfunctional domains, which are not common in eukaryotes. For example, the Lac inhibitor (LacI) and tetracycline repressor (TetR) bind to specific DNA sequence nearby the promoter by oligomerization to make DNA form a loop, and therefore prevent the binding of the transcriptional initiation core elements to the promoter region. Previous studies have shown that in context of regulation of the mammalian genes, placing the LacI binding site downstream of the cytomegalovirus (CMV) promoter or CAG promoter in a synthetic gene circuit inhibits gene expression, despite the efficiency of repression in mammalian expression systems is lower than that in prokaryotic expression systems. Similarly, the dCas9 protein still exhibits weak transcriptional repression function in mammalian system without fusing to any transcriptional repression domain.
The transcription activator-like effector repressor (TALER) protein consists of several “protein modules” in series that specifically recognize DNA, and N-terminal and C-terminal sequences on either side. Each “protein module” contains 33-35 amino acid residues, and the amino acid residues at position 12 and position 13, the key sites for target recognition, are called repeat variable di-residues (RVDs) of amino acid. Each RVD on the TALER protein can recognize only one base. Transcription activator-like effector nuclease (TALEN) is a kind of artificial restriction endonuclease, and is a TALEN fusion protein obtained by the fusion of TALER protein (as a DNA binding domain) with a restriction endonuclease Fok I (as a DNA cleavage domain, also known as a repression domain). TALEN binds to the target site of the genome in cells to form a dimer performing endonuclease activity, which results in DNA's double-strand breaks (DSB) in the spacer regions of TALEN on the left and right sides and thus induces DNA damage repair mechanism. Cells can repair DNA by a non-homologous end-joining (NHEJ) mechanism. NHEJ repair mechanism is not accurate. It is prone to occur errors (deletion/insertion), resulting in frameshift mutation and therefore achieving the purpose of gene knockout.